No abstract

Abstract. The polymerase chain reaction (PCR) was used to develop a test for the detection of Mycoplasma mycoides subspecies mycoides SC in the tissues of animals infected with contagious bovine pleuropneumonia (CBPP). Two sets of primers were designed; one set (MC323/MC358) to amplify a ~1.5-kbp DNA fragment from all the members of the M. mycoides 'Cluster' and the other set (MM450/MM451) specifically amplified a 574-bp DNA fragment from M. mycoides subspecies. The PCR products could be differentiated further by digestion with the restriction enzyme AsnI. Enzyme digestion of amplification products from M. m. mycoides SC produced 2 fragments, whereas the other 2 M. mycoides subspecies, M. m. mycoides LC and M. m. capri, produced 3 fragments. This test was shown to be very sensitive, being able to detect between 10 and 100 organisms. Cattle were experimentally infected with the Gladysdale strain of M. m. mycoides SC, and samples of serum and mucus were taken periodically, as were postmortem samples of lung, lymph node, pleural fluid, synovial fluid, and tracheal swabs. Complement fixation test on serum samples, culture of postmortem tissues, and histopathologic examination confirmed disease. DNA was extracted from postmortem samples and amplified by PCR using primers MM450 and MM451. Digestion of products using AsnI allowed the specific identification of M. m. mycoides SC. This test could confirm CBPP in 48 hours and was thus capable of giving a more rapid result than the traditional methods of culture, isolation, and identification using biochemical and serological techniques.

No abstract

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplusma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M.mycoides subsp, mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp, Capri composed the second group. Mycoplama capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group, Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma, The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.There are six mycoplasmas that make up the group known as the Mycoplasma mycoides cluster. The mycoplasmas in this group are pathogens of cattle, sheep, and goats. M. mycoides subsp. mycoides small colony (SC) is primarily a pathogen of cattle and causes contagious bovine pleuropneumonia (CBPP). M. mycoides subsp. mycoides large colony (LC) is usually found in goats, causing septicemia, arthritis, and pneumonia. M. mycoides subsp. Capri causes pneumonia and arthritis in goats, and Mycoplasma capricolum causes septicemia, arthritis, and mastitis in goats and sheep. Two other groups of unnamed mycoplasmas complete the cluster. These are mycoplasmas represented by the F38 mycoplasma, which is now recognized as the agent of contagious caprine pleuropneumonia (CCPP), and bovine group 7 mycoplasmas, which cause arthritis and mastitis in cattle (8).A number of difficulties have been identified in the classification of this group of organisms. Infrasubspecific varieties of M. mycoides subsp. mycoides (SC and LC forms) have been recognized. The two types are serologically indistinguishable but are quite different in pathogenicity; the SC form causes CBPP in cattle, while the LC form does not. Cottew and Yeats (9) showed that the two types could be separated when they correlated differences in cultural characteristics with differences in heat stability and variation in proteolytic activities. The presence of two enzymes, a-glucosidase and ornithine transcarbamylase, was demonstrated in LC types but not in SC types (17). The ability to produce mycoplasmaemia in mice has also been used to separate LC and SC types (18).M There have also been problems in classifymg the two unnamed mycoplasmas belonging to the cluster. Some field is...